Biocatalytic oxidation using soybean and other legume peroxidases

ABSTRACT

Biocatalytic oxidative processes wherein oxidizable substrates are reacted with peroxides in the presence of a peroxidase such as soybean peroxidase or another legume peroxidase; an oxidative coupling reaction for producing phenolic resins; a method for the purification of peroxidase enzyme-containing extracts generally also are described.

This is a division of application U.S. Pat. No. 07/599,584 filed Oct.18, 1990.

BACKGROUND OF THE INVENTION

The present invention relates to an improved biocatalytic process forthe preparation of phenolic resins using soybean peroxidase and moregenerally to a biocatalytic process for oxidizing phenols and othercompounds using soybean peroxidases.

The present invention further relates to a method for treatingperoxidase enzymes so that they may be used in applications where theuntreated enzyme could not previously be employed More particularly, itrelates to a method for treating a peroxidase enzyme source with apurifying agent to reduce the amount of impurities in the enzyme andthereby reduce contamination in processes using such enzymes as abiocatalyst.

U.S. Pat. No. 4,900,671 commonly assigned to the Mead Corporationdiscloses a method for preparing a phenolic resin which comprisespreparing a solution of a phenol in a water miscible or immisciblesolvent and an aqueous solution of a peroxidase or oxidase enzyme,mixing the two solutions and adding a peroxide or oxygen. The preferredmethod described in this patent makes use of horseradish peroxidase Thereaction is preferably carried out in a mixed solvent system. Theperoxidase is dissolved in water, the phenol is dissolved in a solventwhich may be water miscible or water immiscible. Hydrogen peroxide isadded to the system and reaction occurs on the enzyme. It has now beenfound that soybean peroxidase is much more economical to use in thismethod It has also been found that the quality of resin obtained fromthis method can be improved if the peroxidase is treated as describedherein.

Alberti and Klibanov, BIOLOGICAL DETOXICATION, Chapter 22, Peroxidasefor Removal of Hazardous Aromatics from Industrial Wastewaters, (1982),discloses that phenols can be removed from wastewaters as high molecularweight polymers by the action of peroxidase enzymes. The disclosedmethod relies on the ability of peroxidase enzyme to catalyze, withhydrogen peroxide, the oxidation of a variety of phenols and aromaticamines. Phenolic and aromatic amine free radicals are generated, whichdiffuse from the active center of the enzyme into solution, andpolymerize to polyaromatic products. These high molecular weightpolymers are water-insoluble and can be readily separated by filtrationfrom water.

In the past, peroxidase enzymes have not been available at a cost and ina purity amenable to many biocatalytic processes. For example,horseradish roots, a common source of horseradish peroxidase, arecultivated generally in small quantities and are propagated through rootcuttings, thus making it difficult to scale up production. The limitedavailability of the horseradish root extract coupled with the shortageof alternative sources of enzyme has created a very expensive market forsuch enzymes. Accordingly, there exists in the marketplace a need for anabundant and relatively inexpensive source of peroxidase.

SUMMARY OF THE INVENTION

A principal object of the present invention is to improve thebiocatalytic oxidative process for preparing phenolic resins describedin U.S. Pat. No. 4,900,671 through the use of peroxidases from soybeansand other legumes and plants. It has been found that soybean peroxidasehas better temperature and solvent stability than horseradish peroxidaseand that it is much more economical because it can be obtained fromsoybean hulls which are very inexpensive. This finding suggests thatperoxidase from other sources and particularly legumes may also beadvantageously used in the process.

A more general object of the present invention is to providebiocatalytic oxidative processes using soybean peroxidase or peroxidasesfrom other legumes, rice, and plants (e.g., malvaceous plants such ascotton, see Egley, G. H., et al., Planta 157:224-232 (1983)). One suchoxidative process is oxidative coupling of phenols and aromatic amines,however, there are other oxidative processes in which peroxidases havebeen used in which soybean peroxidase and other plant and legumeperoxidases can be advantageous including wastewater treatment,oxidation of aromatic amines and others.

Still another object of the invention is to provide novel processes forbiocatalytic oxidation wherein oxidation is carried out in the presenceof the hulls of a legume which produces peroxidase, a particularlypreferred legume hull is soybean hulls. It has been found that in manycases it is not necessary to remove peroxidase from the hull byextraction but that the hull can be directly introduced to the reactionmedium where the peroxidase is available as an immobilized enzyme or byin situ extraction to catalyze the oxidative process.

Another object of the invention is to improve biocatalytic processesemploying peroxidases by treating the peroxidase to removenon-peroxidase proteins and other lipophilic materials. Thesecontaminants interfere with many biocatalytic processes by discoloringthe reaction product. They can also lead to emulsification of thereaction system when the reaction is performed in aqueous media makingit difficult to control the molecular weight and molecular weightdistribution of the product and making it difficult to separate theproduct (See Example 2). In accordance with one aspect of the invention,peroxidase as a solution in water, is treated with protein fixatives ordetergents to remove the unwanted materials. In another method, theperoxidase is treated with activated carbon.

Accordingly, one manifestation of the present invention is an improvedprocess of biocatalytic oxidation wherein an oxidizable substrate isreacted with a peroxide in the presence of a peroxidase wherein theperoxidase is a peroxidase of a legume (plants of the familyLeguminoseae) and, more particularly, soybean peroxidase, rice, or aplant of the family Malvaceae.

Another manifestation of the present invention is a process forpreparing a phenolic resin which comprises reacting a phenol withsoybean peroxidase or another of the aforementioned peroxidases in thepresence of a peroxide. In a preferred embodiment of the invention, thisreaction is carried out in a mixed solvent of water and a water miscibleor a water immiscible solvent. In another embodiment of the invention,the peroxidase is supplied to the reaction as soybean or other legumehulls. Other embodiments of the invention utilize peroxidases from riceand malvaceous plants such as cotton.

Another manifestation of the invention is a process for treatingperoxidase to remove proteinaceous or lipophilic contaminants and renderit more useful in biocatalytic processes which comprises preparing asolution of a peroxidase and a protein fixative or a detergent, adding anon-solvent for the peroxidase, re-dissolving the peroxidase andseparating the peroxidase from the contaminants.

In another embodiment of the invention, the peroxidase is purified bymaking a slurry of a peroxidase solution with activated carbon andremoving the activated carbon.

Other objects and advantages will be apparent from the followingdescription and the appended claims.

DEFINITIONS

The term "phenolic resin" as used herein includes phenolic dimers andtrimers as well as higher molecular weight species.

A "unit" of peroxidase means the amount of peroxidase which produces achange of 12 absorbance units measured at 1 cm pathlength in one minuteat 420 nm when added to a solution containing 100 mM potassiumphosphate, 44 mM pyrogallol and 8 mM hydrogen peroxide and having a pHof 6 (Sigma Chemical Co. Peroxidase Bulletin).

DETAILED DESCRIPTION OF THE INVENTION

In the preferred embodiments of the invention, the peroxidase is soybeanperoxidase, however, peroxidases from other legumes are also useful suchas peroxidases from peas, guar beans, garbanzo beans, runner beans andthe non-legume, rice. It is also believed that peroxidases from certainmalvaceous plants such as cotton may be useful. The purificationtechniques described herein, in addition to being effective forperoxidases extracted from legumes, rice and malvaceous plants are alsouseful with horseradish peroxidase, haloperoxidases includingchloroperoxidases, lactoperoxidases, bacterial peroxidases, and fungalligninase.

Peroxidases, being water soluble, are easily harvested by homogenizingthe protein source with water, filtering the homogenate, and retainingthe filtrate.

The filtrate is treated to remove proteinaceous and lipophilicimpurities by adding to the filtrate a solution of a protein fixative ora detergent and forcing the enzyme to precipitate by the addition of anon-solvent for the peroxidase such as acetone or isopropanol. Theprotein fixative and detergent both preferentially render the proteincontaminants insoluble in water. The detergent also insolublizesnonprotein lipophilic impurities. After addition of the fixative ordetergent, a non-solvent for peroxidase is added to the solution toforce the peroxidase and impurities to precipitate. The precipitate isseparated, water is added to redissolve preferentially the peroxidaseand the sample is centrifuged. The peroxidase is recovered as thesupernatant solution While these treatments probably do not completelyremove impurities, they reduce them to a level that the oxidationproduct obtained using the peroxidase is improved in quality.

When using the detergent, the precipitate is preferably treated with asolution of phenol and a small amount of hydrogen peroxide. This appearsto cause the phenol to interact with the detergent and enhance thebinding of the impurities. After about one hour the sample iscentrifuged to remove the impurities. The peroxidase is recovered in thesupernatant solution These processes may be repeated to further purifythe enzyme.

The protein fixatives useful in treating the peroxidase include tannicacid, tannins, monolignols, fulvic acids, lignan, humic acids,melanoidins, proanthcyanidins, stilbenes, depsides, lignin modelcompounds, soluble suberin, flavonoids, soluble lignin, dihydroxyphenylcompounds, kerogen, gallic acid esters, phenolic acids, gallic acidamides, dihydric phenols, hexahydroxydiphenic glucose esters, polymericphenols, bis (hydroxyphenyl) sulfones, bitumens, soluble ligniteextracts, sulfonated phenols and naphthols and their copolymers,melamine/glyoxal/glyoxylate/phenol/naphthol condensates; vegetableextractives, especially rhubarb, mimosa, peat, euphorbia, cassia, rose,tea, grape and saxifragea; sulfonated extractives, especially of mimosawood; and bark extractives, such as oak, eucalyptus, fig, cedar, spruce,pine, walnut, mulberry and chestnut; and graft copolymers derived fromthese extracts Others include synthetic phenolic tanning agents(syntans) such as tanigan, tamol, ledertan, blancotan, basyntan, neosynand nubuctan and phenolic compounds that cause melanization orsclerotization of proteins, especially catechol and dopamine amides,quinones, quinone methides, prenylated phenols and quinones and polymersderived from their oxidation, e.g., melanins and sclerotins, and thelike.

Useful detergents include sodium dodecyl sulfate, sodium caprylate,sodium cholate, sodium decanesulfonic acid, sodium deoxycholate, sodiumglycocholate, sodium deoxyglycocholate, sodium taurocholate, sodiumtaurodeoxycholate, cetylpyidinium chloride, dodecyltrimethyl ammonium,CHAPS, CHAPSO, dioctyl sulfosuccinate, alginic acid. Phenols useful toenhance detergent purification include t-butylphenol and bisphenols suchas bisphenol A.

Removal of the impurities can be enhanced by adding a salt such aspotassium chloride to the aqueous solution of the enzyme in an amount ofabout 1 to 10%. For certain protein fixatives such as the phenols whichare not soluble in water, a small amount of a solvent such as an alcoholmay be used to dissolve these fixatives in water as shown in Examples5-9 below.

Non-solvents of the peroxidase are used to force the enzyme toprecipitate and enable its separation. Useful nonsolvents may be watermiscible or water immiscible, however, they are preferably watermiscible. Representative examples include acetone, isopropanol,n-propanol, methanol, and ethanol.

To purify the enzyme, peroxidase is added to water in an amount of about400 units per ml water. When the protein fixative is used, it isgenerally added to the enzyme solution in an amount of about 1% to 10%based on weight of fixative to volume of enzyme (kg. to 1). Similaramounts of detergent are employed. The volume of the non-solvent whichmust be added to the enzyme solution to separate the enzyme will varywith the nature of the non-solvent but generally 1 to 10 volumes ofnonsolvent per volume of enzyme solution is required.

One oxidative process for preparing phenolic resin in accordance withthe present invention comprises preparing separate solutions of thephenol, enzyme, and peroxide, and mixing them. The phenol is typicallydissolved in an organic solvent, and the enzyme and peroxide aretypically dissolved in water. The solutions may be gradually added to acommon reaction vessel, but in a preferred method solutions of thephenol and the enzyme are pre-mixed and the peroxide solution isgradually added thereto. The enzyme may also be provided on a solidsupport or legume hulls may be used directly. The process may be carriedout on a batch or continuous basis. In any process it is important tolimit the rate of addition of the peroxide since excess peroxide tendsto inhibit the reaction.

The amount of the enzyme used to make the phenolic resin will depend onits activity. The enzyme is not consumed in the reaction but graduallyloses activity during the course of reaction. For practical purposes,the enzyme can be reacted in an amount of about 500 to 500,000 and moretypically 1000 to 5000 units per 100 grams phenol. In other oxidativereactions, analogous amounts of the peroxidase will be used.

The peroxide used is typically hydrogen peroxide, but other peroxidesare also useful. Examples of other potentially useful peroxides includemethyl peroxide, ethyl peroxide, etc.

The peroxide is reacted in an amount of about 0.1 to 2.5 moles per molephenol (or other oxidizable substrate) and, more typically, about 0.1 to1.0 moles per mole phenol. Depending upon the nature of the oxidizingagent, it is reacted neat or as a solution The preferred oxidizingagent, hydrogen peroxide, is dissolved in water. Its concentration mayrange form about 1 mM to 10 M.

Phenols can be reacted in a water-miscible or a waterimmiscible solventRepresentative examples of useful waterimmiscible solvents includehexane, trichloromethane, methyl ethyl ketone, ethyl acetate, andbutanol. Examples of useful water-miscible solvents include ethanol,methanol, dioxane, tetrahydrofuran (THF), dimethyl formamide, methylformate acetone, n-propanol, isopropanol, ethanol, t-butyl alcohol. Thereaction is typically carried out at phenol concentrations of about 1 to100 g per 100 ml solvent.

A number of different procedures may be used to react the phenol orother oxidizable substrate. Solutions of the phenol, enzyme, andperoxide may be individually prepared and metered into a reactionvessel, or solutions of the phenol and enzyme may be pre-mixed and theperoxide gradually added thereto. Alternatively, the enzyme and thephenol may be dissolved in a common solvent and the peroxide addedlater. Those skilled in the art will appreciate that a number ofdifferent reaction/mixing sequences are useful. The peroxide should beadded at a controlled rate which is approximately equal to the rate atwhich it is consumed such that the concentration of the peroxide doesnot build to a level at which it undesirably inhibits the reaction andinactivates the enzyme.

The organic-aqueous system formed upon mixing the phenol, enzyme andperoxide may contain water and an organic solvent in a volumetric ratio(water:organic) in the range of about 1:10 to 10:1, more typically, 1:2to 2:1. The most preferred ratio will vary with the nature of thephenolic monomer(s) that is (are) polymerized.

As indicated earlier, the legume hulls are biocatalytically active andcan be used directly. It is not clear whether the peroxidase is beingextracted by the reaction solvent medium or whether the peroxidasereacts similar to an immobilized enzyme. A combination of bothmechanisms may occur.

The amount of hulls used will depend on their reactivity. To prepare thehulls for the reaction, they are preferably crushed and washed withtoluene and added to ammonium sulfate solution as illustrated in Example14 below. Aged hulls may work as well as fresh ones. The reaction may becarried out by simply preparing a slurry of the hulls in an aqueoussolution of the phenol and gradually adding peroxide thereto at acontrolled rate which does not result in reaction inhibition.Alternatively, the hulls can be packed in a column and peroxide and theoxidizable substrate passed over them to yield the oxidized product.

Variations in the way the hulls are prepared produce color developerresins with either low or high natural color. Hulls added to 840 ml 0.2to 0.4M ammonium sulfate or sodium sulfate produced much lighter resin.Hulls washed with toluene then ethyl acetate and added to ammonium orsodium sulfate solution produced still lighter resin. These sameobservations were made with soluble soybean peroxidase andsolvent-washed hulls. The use of solvent-washed hulls and polymerizationwith the enzyme in 0.4M ammonium sulfate produces low-color resins. Thesulfate presumably reduces color by salting-in the colored impurities inthe enzyme, preventing their release to the organic phase containing thepolymer. Washing the hulls removes many of these color-causingimpurities prior to the reaction.

The additional thermal stability of peroxidase from soybean hulls wasdemonstrated by heating soybeans at 90° C. for 30 minutes. Peroxidaseactivity measured following extraction from the resulting hulls was thesame per gram hull in the 90° C. treated sample as the untreatedcontrol. Further, polymerization of bisphenol A in 45% n-propanol withsoybean peroxidase produces a low-monomer color developer up to 45° C.reaction temperature at 50 units per gram monomer. The same reactionwith Finnsugar or Sigma horseradish peroxidase succeeds only up to 20°C. Further, the reaction with soybean peroxidase succeeds at one-fourththe activity level required with horseradish peroxidase at 25° C.

Reaction temperature will vary with the substrate and the enzyme, mostenzymes are temperature sensitive and a temperature should be selectedwhich does not inhibit the reaction.

The reaction of the phenol proceeds at room temperature, buttemperatures of about 0° to 70° C. can be used. The enzymes aretemperature sensitive and can lose their activity if the reactiontemperature becomes too high. However, some latitude exists, dependingupon the solvent system which is used. Certain solvents can stabilizethe enzyme and thereby permit the use of higher temperatures. There isevidence in the literature that temperatures up to 100° C. may be usefulwith some peroxidases.

The activity of peroxidases is pH dependent. The oxidative reactions aretypically carried out at a pH in the range of 4 to 12 and, preferably, 4to 9, and, more preferably, about 6. A pH may be selected at which theenzyme is highly active. This will vary with the nature of the enzymeand its source. Buffers can be used to maintain pH, but are not usuallyrequired. One example of a useful buffer is a potassium phosphatebuffer.

While reference is herein made to the bulk pH of the reaction system,those skilled in the art will appreciate that it is the pH in themicro-environment of the enzyme that is critical. Thus, where the phenolis dissolved in a water immiscible solvent and the enzyme solution isdispersed in the solution of the phenol, it is the pH of the enzymesolution which is critical.

Phenolic resins prepared in accordance with the present invention areuseful in a variety of applications depending on the nature of thephenol and the molecular weight distribution of the resin. The resinsare often mixtures of dimers, trimers, and higher molecular weightoligomers.

The molecular weight of the phenolic resin can be adjusted dependingupon its particular end use. In one embodiment, the process of thepresent invention provides a phenolic resin which is useful as adeveloper in recording materials such as carbonless copy paper,heat-sensitive recording paper, electrothermographic recording paper andthe like. The phenols used in developer resins are preferablypara-substituted. Developer resins may range from about 500 to 5000 inmolecular weight.

In another embodiment, the process of the present invention provides aphenolic resin which is useful as an adhesive. The phenols used inadhesives need not be parasubstituted. The resins typically range fromabout 1000 to 15,000 in molecular weight but molecular weights up to atleast 30,000 are attainable. Among other factors affecting molecularweight are solvent selection, phenol selection, and reaction conditions.

Phenols which are preferred for reaction in the present invention arerepresented by the Formula (I): ##STR1## wherein Y and Z are selectedfrom the group consisting of a hydrogen atom, a halogen atom, an alkylgroup, an alkoxy group, an aryl group, an allyl group, a phenylalkylgroup, a --COOR group, a --NR¹ R² group, where R represents a hydrogenatom or a lower alkyl group, and R¹ and R² represent a hydrogen atom, analkyl group, or a phenylalkyl group or Z in conjunction with theadjacent meta position forms a condensed benzene ring. Sincepolymerization proceeds via the ortho or para positions, when Y is atthe ortho or para position, at least one of Y and Z must be a hydrogenatom or Z must form said condensed benzene ring. Y is preferably para tothe phenolic hydroxyl group. Otherwise, the phenol adds as a terminalgroup as discussed below. At the para position, long chain alkyl groupshave a tendency to slow the reaction. The reaction appears to proceedbest when Y is p-phenyl, p-methoxy or p-halogen.

A single phenol or a mixture of phenols may be used in the process ofthe present invention. In certain applications it may be desirable toproduce phenolic resins having certain terminal groups. This can beaccomplished by reacting phenols in which the Y substituent is in thepara position and Z is not a condensed ring with other phenols in whichat least one of Y and Z is a hydrogen atom or Z is a condensed ring toprovide copolymers. In this case the resin contains the Z substituent asa terminal group. When the para position is unsubstituted,polymerization proceeds via the ortho and/or para position andZ-substituted phenols can be incorporated mid-chain.

The alkyl group represented by Y and Z may contain up to 10 carbon atomsand include such alkyl groups as t-butyl, n-butyl, octyl, nonyl, etc.When R, R₁, and R₂ represent an alkyl group, it is typically a loweralkyl group having 1 to 4 carbon atoms.

Representative examples of alkoxy groups for Y and/or Z have 1 to 10carbon atoms and include methoxy and ethoxy. When Y or Z is an arylgroup, it is typically a phenyl group or substituted phenyl group suchas a halogen-substituted phenyl group, an alkyl-substituted phenyl or aphenol group such as a 4'-phenol group.

Examples of a halogen atom include fluorine, chlorine, bromine andiodine.

Representative examples of phenylalkyl groups include benzyl,isopropylidene phenyl, butylidene phenyl, isopropylidene-4'-phenol, andbutylidene-4'-phenol.

Specific examples of phenols which can be polymerized in accordance withthe process of the present invention are phenol, 4-t-butylphenol,4-n-butylphenol, 4-ethylphenol, cresol, p-phenylphenol, p-octylphenol,p-nonylphenol, p-hydroxybenzoic acid, 4-hydroxynaphthoic acid,p,p'-biphenol, 4-aminosalicylic acid, salicylic acid, methyl salicylate,ethyl salicylate, 4,4'-isopropylidenediphenol, ethyl 4-hydroxybenzoate,etc.

In one embodiment, a phenolic developer resin capable of reacting withan electron-donating color precursor and producing a visible image isrepresented by the formula (II): ##STR2## where n is greater than 2, thephenolic units of the resin are directly bonded to one another throughpositions ortho or para to the hydroxyl group, Y is not hydrogen and ispresent at a position meta or para (preferably para) to the hydroxylgroup.

In accordance with another embodiment, the phenolic developer resin isrepresented by the formula (III): ##STR3## where n, Y, and Z are definedas in formula (II).

The phenolic resins can be homopolymers or copolymers, i.e., theindividual Y or Z groups in a given phenolic developer resin may be thesame or different and the Y groups may be located at different positionsin accordance with the formula (II).

The phenolic developer resins may be metal-modified in a manneranalogous to novolak developer resins to improve their reaction withcolor precursors and thereby improve the density and fastness of theimage. For example, the phenolic developer resins can be modified byreaction with a salt of a metal selected from the group consisting ofcopper, zinc, cadmium, aluminum, indium, tin, chromium, cobalt, andnickel.

This modification can be made in an otherwise known manner. One methodis by mixing and melting the resin with an alkanoate salt such as zincpropionate, zinc acetate, or zinc formate in the presence of an ammoniumcompound such as ammonium carbonate or ammonium acetate. The practicedescribed in U.S. Pat. No. 4,173,684 can also be used.

The zinc-modified phenolic developer resins can also be formed byreacting zinc oxide or zinc carbonate and ammonium benzoate or ammoniumformate with the resins in a manner analogous to the teachings in U.S.Pat. Nos. 4,165,102 and 4,165,103. Alternatively, the zinc-modifiedphenolic developer resins can be prepared by reaction with zinc chlorideas shown in the examples below.

The metal content of the metal-modified phenolic developer resins shouldbe more than 0.5 percent by weight and may range up to 25% by weight.Usually, a range of about 1.5 to 5 percent by weight is used.

In addition to chemically modifying the phenolic developer resins asdescribed above, other means conventionally used in the art to improvethe developing ability of phenolic developer resins, can be used inconjunction with the phenolic developer resins of the present invention.For example, acidic metal salts can be incorporated into coatings of thephenolic developer resins as described in U.S. Pat. Nos. 3,516,845 and3,723,156. The phenolic developer resins of the present invention canalso be used in combination with other phenolic developer resins orcompounds and need not be used alone.

Recording materials utilizing phenolic developer resins to producecolored images from colorless or substantially colorless materials arewell known. Specific examples of such recording materials includepressure-sensitive carbonless copying paper, heat-sensitive recordingpaper, electrothermographic recording paper, and the like. They aredescribed in more detail in U.S. Pat. Nos. 2,712,507; 2,730,456;2,730,457; 3,418,250; 3,432,327; 3,981,821; 3,993,831; 3,996,156;3,996,405 and 4,000,087, etc. A photographic material has been developedwhich utilizes this method for forming colored images. See, for example,U.S. Pat. Nos. 4,399,209 and 4,440,846 to The Mead Corporation.

Recording materials can be prepared in a conventional manner. To providea developer sheet, the phenolic developer resin may be dissolved in anappropriate solvent (typically acetone) and applied to the surface ofthe paper by blade or roll coating or the like. Alternatively, thedeveloper resin may be used in the form of a resin grind analogous tothe resin grinds described in U.S. Pat. No. 3,924,027 to Saito et al.For example, the resin may be pulverized and mixed with an organic highmolecular compound such as starch or styrenebutadiene latex. Thismixture is dispersed in water or a solvent that does not readilydissolve the phenolic developer resin or the high molecular compound andcoated on an appropriate support.

The developer resin is usually applied in an amount of about 0.2 to 0.4lbs. or resin/1300 sq. ft. (solids).

Where a self-contained recording material is desired, a mixture of thephenolic developer resin and microcapsules containing the developer canbe coated upon a support as one layer, or the developer and themicrocapsules can be applied in separate layers. For the preparation ofphotosensitive recording materials, see U.S. Pat. Nos. 4,399,209 and4,440,846 which are incorporated herein by reference.

In addition to being useful as developer resins and as adhesives,phenolic resin products are useful in other applications. In particular,the lack of a methylene bridge imparts advantageous properties to theresins as counterparts. The resins should exhibit greater resistance tophotolytic and thermal degradation, greater rigidity and greaterconductivity making the resins potentially attractive for plasmaresistance in photoresists, as conductive polymers, antioxidants forplastics, rubbers and the like, and as molding materials for hightemperature applications. The higher density of functional hydroxygroups is being investigated for use in epoxy resin systems where highercrosslink densities should yield higher thermal deformationtemperatures.

The resins produced in the present invention are also useful incomposites analogous to epoxy resins. They are particularly useful infrictional composites such as brake linings, transmission bands,structural composites.

The process of the present invention is also useful in preparing lowermolecular weight compounds such as dimers or trimers. Accordingly, informula (II) and (III) above, the process should be useful in preparingcompounds for which n is 1 or 2 as well as higher molecular weightcompounds in which n is greater than 2.

In addition to being useful in preparing phenolic resins, soybeanperoxidase and peroxidases from other legumes, rice and malvaceousplants are also believed to be useful in the following reactions:oxidative coupling of aromatic amines and indoles; oxidation of anionsto free radicals (e.g., carboxlyates, cyanide, thiolates, sulfite,ascorbate); oxidation of metallic mercury; hydroxylation of aromaticphenols and amines; oxidation of anions to inium ions (e.g., iodide,thiocyanate); oxidation of phenols to quinone methides and aromaticamines to imines; formation of disulfides from thiols, sulfoxides fromsulfides and halides; formation of superoxide from thiols and oxygen;dehalogenation of halogenated phenols and aromatic amines;depolymerization of lignin and coal; iodination of aromatics; cleavageof uricates and sugars; oxidation of olefins to alcohols; cleavage ofaldehydes to acids and ketones; demethylation of N-substituted aromaticamines; and treatment of wastewater for contaminants phenols and/oraromatic amines (see Alberti and Klibanov, supra).

In addition to being useful in the reactions discussed above, soybeanperoxidase and hulls harvested and treated using the techniquesdescribed herein may also be useful in other applications previouslythought to require the use of more expensive enzymes including thefollowing: as biocides in pulp and paper mill streams (see U.S. Pat. No.4,478,363); in enzymatic bleaching of Kraft pulp (see Intl publ No WO87/00564, Intl Appln No PCT/US86/01476, Eriksson, KE, Chem Abstr112:219038h, Ander, P., Chem Abstr 112:212969d); as a catalyst inimmobilization of leachable toxic soil pollutants (see Shannon, M.J.R.,et al. Appl. Env. Microbiol (1988) 54:1719-1723); in medical diagnosticsin coupling to antibodies and detection with leuco-dyes (see U.S. Pat.Nos. 3,694,207; 4,828,983; 4,778,753; EP 218,083); in quinone dyesynthesis (see Czch pat CS 247,596 Bl); accelerated drying of lacquers(see Japn. Pat. 01163272); in synthesis of melanin-like dyes (see U.S.Pat. No. 4,609,544); in oxidative thickening of pectins (see U.S. Pat.No. 4,672,034); as bioamperometric sensors for phenol detection (seeBonakdar, M., Chem Abstr 112:90986j); in analytical determinationperoxides (see Berlin, P., Chem Abstr 112:73352); as a bacteriacide toprevent tooth decay (see Grisham, M.B., Chem Abstr 112:117169j, Kessler,U.S. Pat. No. 4,476,108); in inactivation of mutagenic substances (seeChem Abstr 93:21203d, Japanese Patent 55037180); in treatments andcompositions to promote wound healing (see U.S. Pat No. 4,503,037); inwaste water treatment (see U.S. Pat No. 4,623,465, Science 221:259-261(1983), Enzyme Microb Technol 3:119-122 (1981), Davis, S., Chem Abstr112:222683v, Alberti and Klibanor, supra), as a preventative forartherosclerosis (see Khanin, AL Chem Abstr 82:68359x); in activation ofcommercial enzymes (see Tressel, P., BBRC 92:781-786 (1980)); inquantitation and detection of gums (see Dickmann, R.S., Chem Abstr111:55931k); in bleaching of fabrics (see Kirk, O., Chem Abstr112:101222k); in stabilization or removal of phenols in beer (seeGiovanelli, G., Chem Abstr 112:156624y); and in solubilization of coal(see Scott, C.D., Chem Abstr 113:26681z).

Of the foregoing applications, the soybean enzyme and hulls areparticularly useful in wastewater treatment where they can besubstituted for horseradish peroxidase.

The invention is illustrated in more detail by the followingnon-limiting examples.

EXAMPLE 1

Purification and use of horseradish peroxidase 100 g of a Tannic Acidsolution was dissolved in 900 ml of 0.1 M phosphate buffer of pH=6 toproduce a 10% solution (W/V). The volume of the solution was adjustedwith the phosphate buffer to 1000 ml. 200 ml of horseradish peroxidasesolution from Finnsigar Biochemicals was stirred at room temperature and20 ml of the 10% tannic acid solution was added over 5 minutes. Themixture was stirred for an additional -5 minutes and then poured into800 ml of acetone with stirring. After 5 minutes the solution wasfiltered through a Whatman #4 paper filter. The precipitate wasdissolved in 200 ml of water and centrifuged at 1500 Xg for 30 minutes.Following centrifugation, the supernatant was used as the source ofenzyme in a biocatalytic process for preparing phenolic resin. At theend of the polymerization reaction, aqueous and organic phases separatedspontaneously. The organic phase was filtered through diatomaceous earthto remove particles and then evaporated with steam to yield a lightcolored amber phenolic resin.

EXAMPLE 2 (COMPARISON)

The procedure of Example 1 was repeated except that the horseradishperoxidase was not treated with tannic acid prior to its use as aperoxidase enzyme in the polymerization reaction. At the end of thereaction a stable emulsion prevented separation of the aqueous andorganic phases. The phenolic resin recovered from this reaction produceda red-brown resin. The phenolic resins produced in examples 1 and 2 wereanalyzed by HF gel-permeation and reverse phase chromatography whichshowed that the resins were very similar except for color.

EXAMPLE 3

Method of Harvesting Soybean Peroxidase: One kg of dry soybeans obtainedfrom J.R. Kelly Company, Collinsville, Ill. was placed in a blender andhomogenized in 5 1 of water. The homogenate was filtered through fourlayers of cheesecloth and the filtrate saved. To 500 ml of the filtratewas added 75 ml of 10% tannic acid in 0.1 M phosphate buffer. Themixture was centrifuged at 1500 Xg for 30 minutes and the supernatantsaved. Protein in the supernatant was precipitated by pouring thesupernatant into 3 volumes of acetone at room temperature. The acetonewas decanted and the precipitate was dissolved in 500 ml of water.Protein contaminants were further removed by the addition of 2.5 ml of50% ZnCl₂ in water. The Zn treated protein was centrifuged at 1500 Xgfor 30 minutes and the supernatant was decanted. The supernatant waspoured into 3 volumes of acetone to precipitate the Zn-treated protein.The acetone was decanted and the precipitate was dissolved in 100 ml ofwater and used as the source of soybean peroxidase enzyme.

Biocatalytic Polymerization of 4,4'-isopropylidenediphenol (BisphenolA): The soybean peroxidase enzyme obtained above is employed as abiocatalyst in a normal polymerization of bisphenol A except that only70% of the normal amount of enzyme is used (normal enzyme is fromhorseradish roots). 100 g of bisphenol A is dissolved in 60 ml ofacetone and 140 ml of ethyl acetate. 3500 units of the soybeanperoxidase at 14 units/mg protein (0.25g) are dissolved in 400 ml ofdeionized water. Both solutions are added to a one liter, three-neckedround bottom flask and stirred at 300 rpm's. 67 ml of a 15% hydrogenperoxide solution is added over an approximately 6 hour period. Uponcompletion of the reaction, phase separation occurs spontaneously andthe product is easily recovered from the organic phase by evaporation.The product shows the same distribution of polymer and sameglass-transition temp Tg=83° C. as obtained when using horseradishperoxidase and bisphenol A except that no residual monomer is detected.Thus, using 2/3 the normal amount of enzyme, a 100% yield of bisphenol Apolymer is obtained using the pre-treated soybean peroxidase.

EXAMPLE 4

A volume of a detergent (sodium dodecyl sulfate) is added to a solutionof horseradish peroxidase to give a 2% final concentration of detergent(W/V). The protein is precipitated by pouring the mixture into 3 volumesof isopropanol. The precipitate is dissolved in a minimal volume ofwater and assayed for peroxidase. The treated peroxidase is mixed withbisphenol A dissolved in 20% acetone in the ratio of 50 units peroxidaseper gram of bisphenol A per 6 ml of 20% acetone. A 15% hydrogen peroxidesolution was added in the ratio of 0.18 ml per g of bisphenol A over aperiod of one hour. After one hour the mixture was centrifuged at 1500Xg for 15 minutes and the bisphenol A resin recovered. The supernatentwas used as the source of enzyme in a normal polymerization reaction.

EXAMPLE 5

Soybeans are cracked in a grinding mill, extracted with acetone, andsoaked in water to loosen the hulls. The hulls float to the surface andare isolated by pouring them onto a screen. The hulls are homogenized in0.4 M ammonium sulfate filtered through cheese cloth and the homogenateis separated into three samples which were treated as follows:

Sample 1: The homogenate is adjusted to 30% isopropanol (V/V), andslurried with activated carbon at 1% W/V concentration. The slurry isstirred for a few minutes after which the mixture is filtered throughcelite on a Whatman GF/F glass fiber filter (0.7 micron pore size). Thefiltrate is used as a source of purified soybean peroxidase.

Sample 2: The homogenate is slurried in water with 0.3% activatedcarbon. The slurry is stirred, filtered through celite and used as asource of purified soybean peroxidase.

Sample 3: The homogenate 20% acetone (W/V) and slurried with 1%activated carbon (W/V). The mixture was filtered through celite. Theactivated carbon was washed with 30% isopropanol in water (W/V) and theisopropanol wash was used as a source of purified soybean peroxidase.

In each of the three procedures, significant amounts of impurities areremoved from the soybean peroxidase in an economical manner to allow theuse of the purified enzyme in a wide variety of applications where theseimpurities might otherwise interfere.

EXAMPLE 6

Soybean seed hull extract was mixed with an equal volume of 20% t-butylphenol in isopropanol. 0.5 volumes of water was added and the mixturecentrifuged at 1500 Xg for 15 minutes. The aqueous layer was removed bysiphoning and adjusted to 6-10% KCl solution W/V with solid potassiumchloride. The treated extract was poured into 4 volumes of acetone. Theacetone solution was centrifuged and decanted. The precipitate wasdissolved in water to yield purified soybean peroxidase at 4M KClconcentration. The treated soybean peroxidase was used in thepolymerization of bisphenol A to produce a low molecular weight polymerof bisphenol A.

EXAMPLE 7

The procedure of Example 6 was repeated using t-butyl phenol as thepurifying agent in n-propanol to provide purified soybean peroxidase.

EXAMPLE 8

The procedure of Example 6 was repeated using bisphenol A as thepurifying agent in isopropanol to provide purified soybean peroxidase.

EXAMPLE 9

The procedure of Example 6 was repeated using bisphenol A as thepurifying agent in n-propanol to provide purified soybean peroxidase.

In each of Examples 7-9, the soybean peroxidase enzyme is used in thepolymerization of bisphenol A to provide a bisphenol A resin similar tothat obtained in Example 6 and 10. Examples 6-9 are useful with hullsisolated from aged beans (over 1 yr. old) or hulls contaminated withsignificant amounts of bean material as well as fresh hulls.

EXAMPLE 10

Horseradish root extract was mixed with an equal volume of 20% t-butylphenol in isopropanol. 0.5 Volumes of water was added and the mixturecentrifuged at 1500 Xg for 15 minutes. The aqueous layer was removed bysiphoning and mixed with about 6-10% KCl solution. The treated extractwas poured into 4 volumes of acetone. The acetone solution wascentrifuged and decanted. The precipitate was dissolved in water toyield purified horseradish peroxidase. The treated horseradishperoxidase was used in the polymerization of bisphenol A to produce alow molecular weight polymer of bisphenol A.

EXAMPLE 11

The procedure of Example 10 was repeated using t-butyl phenol as thepurifying agent in n-propanol to provide purified horseradishperoxidase.

EXAMPLE 12

The procedure of Example 10 was repeated using bisphenol A as thepurifying agent in isopropanol to provide purified horseradishperoxidase.

EXAMPLE 13

The procedure of Example 10 was repeated using bisphenol A as thepurifying agent in n-propanol to provide purified horseradishperoxidase.

In each of Examples 11-13, the horseradish peroxidase enzyme is used inthe polymerization of bisphenol A to provide a bisphenol A resin similarto that obtained in Examples 5 and 9.

EXAMPLE 14

The economic and practical utility of soybean seed hulls was tested as asubstitute catalyst in the synthesis of bisphenol A polymer, awell-established color developer which can be made in a predicablemanner using free horseradish or soybean peroxidase in solution. Groundsoybean hulls were obtained from Cargill, Inc. of Sidney, Ohio. Thehulls were screened through a 30 mesh screen and the -30 mesh hulls wereused in the following examples. The reaction conditions were 100g hullsadded to 840 ml aqueous solution and mixed with 200g bisphenol Adissolved in 360 ml n-propanol. Fifteen percent hydrogen peroxide wasadded gradually over 2.5 hours until 60 mole percent was added. Noperoxide excess was indicated by using starch-iodide test strips duringthe reactions. Stirring was at 300 rpm and the exothermic polymerizationwas allowed to proceed without temperature control. Typically, thetemperature may reach a maximum of 40° C. At the end of the reaction,the mixture was centrifuged at 1,500xg for 10 minutes and the aqueoussupernatant decanted. Occasionally, a 2-fold dilution with water isrequired for good separation on centrifugation. The polymeric resin andhulls were stirred with 1 liter ethyl acetate, recentrifuged and thesupernatant filtered through a Whatman GF/F Glass fiber filter. Theethyl acetate was separated from the polymer by evaporation on a steambath followed by a hot plate. The yield of resin was 60-90% of thestarting monomer.

Having described the invention in detail and by reference to preferredembodiments thereof, it will be apparent that modifications andvariations are possible without departing from the scope of theinvention defined in the appended claims.

What is claimed is:
 1. A method for the purification of a peroxidaseenzyme comprising forming a solution of a peroxidase enzyme and aprotein fixative selected from the group consisting of tannic acid,tannins, monolignols, fulvic acids, lignan, humic acids, melanoidins,proanthcyanidins, stilbenes, depsides, lignin model compounds, solublesuberin, flavonoids, dihydroxyphenyl compounds, kerogen, gallic acidesters, phenolic acids, gallic acid amides, hexahydroxydiphenic glucoseestes, polymeric phenols, bis (hydroxyphenyl) sulfones, bitumens,soluble lignite extracts, sulfonated phenols and naphthols and theircopolymers, melamineglyoxal/glyoxylate-phenol/naphthol condensates; peateuphorbia, cassia, rose, tea, grape and saxifragea; sulfonated extracts,and bark extracts, and graft copolymers derived from said extracts andsyntans; adding a non-solvent for said enzyme to said solution to causesaid enzyme to precipitate, and redissolving said enzyme and theperoxidase is recovered.
 2. The method of claim 1 wherein saidperoxidase enzyme is a soybean peroxidase.
 3. The method of claim 2wherein said protein fixative is tannic acid.